A DNA Pairing-enhanced Conformation of Bacterial RecA Proteins

Nami Haruta,Xiong Yu,Shixin Yang,Edward H Egelman,Michael M Cox

doi:10.1074/jbc.m308563200

Abstract

The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs. The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB). In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps. This same trend is seen with the EcRecA protein. The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P. The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA. The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A. The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work. 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state. 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain.

Highlights

The RecA protein of Escherichia coli (EcRecA)1 is the prototype of a class of proteins playing a central role in the recombinational DNA repair in all organisms
The fourstrand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec singlestranded DNA-binding protein (SSB)
1) When a segment of a RecA filament bound to single-stranded DNA (ssDNA) is forced into the P state, the segment becomes “pairing enhanced.” 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state

Summary

Introduction

The RecA protein of Escherichia coli (EcRecA) is the prototype of a class of proteins playing a central role in the recombinational DNA repair in all organisms. The single-stranded DNA-binding protein (EcSSB) has a major effect on all RecA reactions, playing a role in at least two steps in DNA strand exchange. The stimulatory effects of SSB can be partially compensated for by forming RecA filaments on ssDNA under conditions in which little secondary structure exists, and adding the Mg2ϩ needed for efficient reaction (a Mg2ϩ-shift protocol) [17, 18]. The DrRecA protein is similar to other bacterial RecA proteins It forms helical filaments on DNA, hydrolyzes ATP and dATP, and promotes DNA strand exchange [26]. The most substantial difference documented to date concerns the pathway for DNA strand exchange, where the order of substrate binding changes in reactions promoted by the DrRecA protein.

Results

Discussion

Conclusion